Morphogenesis nicosia betting
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Moreover, this is independent of genomic features, such as DNA repeating elements and recombination rate. In particular, we confirmed that oncogenes are preferentially amplified and tumour suppressors are preferentially deleted. To investigate their functional impact, we measured the gene expression changes upon copy number variation.
We observed that amplification of a gene leads to its higher expression whereas deletion results in decreased gene expression, which suggests that amplifications activate dominant genes and deletions inactivate recessive genes. The two classes of cancer genes are vastly modified consistent with their functional roles as oncogenes and tumour suppressors, with the few exceptions of frequently amplified recessive genes underlying complex epigenetic regulation. The mutational spectrum of the human genes in cancer, together with their systems level properties, can be exploited to identify novel targets for anti-cancer therapy, in which synthetic lethality emerges as a promising approach.
Based on the working hypothesis that paralogous genes may engage in synthetic lethal interactions due to the functional redundancy between them, we combined several gene properties to predict synthetic lethality between paralogous gene pairs. Out of 37 candidate gene pairs, we experimentally validated the synthetic lethal interaction between two components of the cohesin complex, STAG1 and STAG2.
NCG 5. NCG has been increasingly appreciated as a central resource for cancer genomics research, facilitating candidate prioritization for hypothesis testing and experimental planning in a wide range of studies. Francesca Angiolini The adhesion molecule L1: a novel player in ovarian cancer vasculature Ovarian cancer OC represents an outstanding clinical challenge because of its high mortality rate, due to tumor relapse and chemoresistance.
The identification of novel strategies for the treatment of OC is clearly an unmet clinical need. In this context, drugs interfering with tumor neovascularization have shown promising results in clinical trials, however, with only transient beneficial effects.
Thus, the definition of novel druggable targets within OC vasculature will likely contribute to improve the clinical management of patients. L1 is a transmembrane adhesion molecule in the nervous system. However, several studies have demonstrated its involvement in different types of human cancer. In this context, L1 expression is generally associated with poor diagnosis, aggressive behavior and increased metastatic spread. Our laboratory has obtained compelling evidence that L1 is aberrantly expressed in tumor vasculature and exerts a pleiotropic function in endothelial cells.
Based on these findings and on the pivotal role of angiogenesis in ovarian cancer, in this work I have investigate the functional role of L1 within the OC-associated vasculature. My results revealed that L1 is abundant in OC vasculature as compared to normal vessels and it was found to be a causal player in OC progression.
This research, besides giving insights into novel pathways involved in pathological angiogenesis, provides the rationale for exploring the clinical relevance of L1 expression and function in OC vessels and in their crosstalk with tumor cells, possibly opening new avenues for the development of innovative targeted therapies for OC malignancy.
Marco Annoni Lie to Me. The Ethics of Truth-telling and Deception for Oncology This dissertation deals with the ethics of truth telling and deception in medicine. Should clinicians tell the truth, even if the truth may cause patients irremediable psychological and physical harm? Are lies told with a benevolent intent always blameworthy? Are deception and concealment less culpable than lying? How can physicians determine, between two alternative verbal disclosures, which one provides the optimal balance between truthfulness and beneficence?
In this dissertation I endeavor to elaborate an answer to these and other questions by setting forth a normative theory of truth telling and benevolent deception for medical professionals, and especially for those operating in clinical oncology. Throughout this work I defend two main ideas. First, clinicians have a duty of veracity in all their professional communications, but in exceptional cases other considerations of beneficence and compassion may override this prima facie obligation.
Second, clinicians may resort to clinical deception only if they have ruled out all other truthful courses of action and would be ready to hypothetically defend and actually disclose their behavior in public. This view, I contend, has several advantages over competing accounts and provides clinicians with a practical way of approaching moral dilemmas about truth telling and deception in clinical medicine.
Sina Atashpaz A cell reprogramming-based approach to study 7q Although both these diseases share common features such as intellectual disability and craniofacial dysmorphism, they can be distinguished by distinct social and language abilities: WBS patients characterized by hypersociality and comparatively well-preserved language skills while 7dup-ASD is associated with impairment in social interaction and communicative skills.
The involvement of same genetic interval in these disease, points out to small subset of dosage-sensitive genes affecting cognition, social behavior and communication skills. Among the genes in the deleted region, some were shown to contribute to the abnormalities in these patients through transgenic mice models and individual case reports. However, the precise cellular and molecular phenotypes associated with these syndromes in disease-relevant cell-types are unknown due to the scarce availability of primary diseased tissues.
During my PhD studies, I focused on the functional dissection of these complementary diseases at the level of transcriptional deregulation in patient-derived iPSC and its differentiated derivatives such as neural crest stem cells, mesenchymal stem cells, and neural progenitors. To this end, we have assembled a unique cohort of typical WBS, atypical WBS patient with a partial deletion and 7dup-ASD patients along with unaffected relatives , and then I used mRNA reprogramming to establish and characterize at least 3 independent iPSC lines from a total of 12 individuals.
High throughput mRNA sequencing on iPSC revealed critical transcriptional derangements in disease-relevant pathways already at the pluripotent state. These alterations found to be selectively amplified upon differentiation into disease-relevant lineages, thereby establishing the value of large iPSC cohorts in the elucidation of disease-relevant developmental pathways.
Finally, we created an open-access web-based platform to make accessible our multi-layered datasets and integrate contributions by the entire community working on the molecular dissection of the 7q Cells have developed numerous mechanisms to overcome replication stress RS. After the loss of key factors, that help to deal with RS, deleterious events can occur. When DNA replication is blocked, checkpoint activation ensures structural stability of the replisome avoiding fork collapse and promoting DNA replication resumption.
We identified one possible source of ssDNA gap accumulation at forks by showing that homologous recombination protein Rad51 is required for optimal function of Pola at stressed replication forks. RadPola interaction is likely to be important for stalled fork resumption. Overall this provides structural and molecular insights into the metabolisms of replication forks under stressful conditions.
While clathrin-mediated endocytosis destines EGFR for recycling and signaling, internalization through non-clathrin endocytosis NCE targets the receptor for degradation. Since NCE appears to be a major negative regulator of EGFR levels, a more complete picture of this pathway would likely reveal new insights into aberrant EGFR signaling observed in many types of cancer.
By combining a candidate gene approach with an unbiased proteomic approach, we have defined EGFR-NCE as molecularly distinct from other NCE pathways, relying on functional regulators not previously implicated in endocytosis. Iros Barozzi Overlapping sequence features of mammalian enhancers coordinately control engagement of transcription factor consensus sites and nucleosomal occupancy In mammalian cells transcription factors TFs bind only to a small fraction of the available consensus sites in the genome.
In particular, they prefer sites embedded in regions of computationally predicted high nucleosomal occupancy. This is compatible with non-exclusive mechanisms of nucleosome-driven TF-binding and nucleosome-mediated masking of TF binding sites, suggesting that TFs, and in particular pioneers, must overcome a strong barrier in order to engage binding. Exploiting the available information for the hematopoietic master regulator Pu.
We identified a minimal set of features, which predicts Pu. Consistent with this, while Pu. As predicted, engaged consensus sites showed higher sequence-encoded nucleosome occupancy compared to the myriad of non-occupied and likely non-functional consensus sites that randomly occur in mammalian genomes. These data reveal a basic organizational principle of mammalian enhancers whereby TF-engagement at its consensus sites and nucleosome occupancy are coordinately controlled by overlapping sequence features.
This model also suggests that co-evolution of these features may be crucial to ensure cell-type specific enhancer activation. The nucleosomal patterns at Pu. Federica Basilico A biochemical and structural study of the kinetochore - centromere interface Faithful chromosome segregation during mitosis requires the dynamic interaction between spindle microtubules and kinetochores, multiprotein complexes built on centromeres.
A group of kinetochore proteins associates with centromeres throughout the cell cycle and is thus named constitutive centromere-associated network CCAN. Biochemical and functional analyses indicate that CCAN proteins are organized in sub-complexes. However, the exact organization of these sub-complexes has not been fully elucidated to date.
The aim of my project has been the biochemical reconstitution of CCAN sub-complexes and their structural and functional characterization. In particular, this dissertation dwells upon the results I have obtained regarding three different but intrinsically related topics.
The first steps towards an in vivo validation of these results are also described. Third, I have been involved in establishing in the laboratory techniques for the in vitro reconstitution of recombinant nucleosomes. The production of material of good quality and quantity has recently been achieved, supporting the analysis of in vitro interactions between nucleosomes and kinetochore components.
Specifically, I illustrate some preliminary observations concerning a direct interaction of Mis12 complex with nucleosomes. A-T is a rare autosomal recessive neurodegenerative disease characterized by neurological dysfunction, multisystem abnormalities and cancer predisposition. Recent observations are expanding the roles of these kinases outside the nucleus and several proteomic screens have identified hundreds of cytoplasmic substrates phosphorylated by ATM and ATR.
Our group previously showed that ATR is activated by mechanical stress and localizes to the nuclear envelope; moreover, recent unpublished observations in our lab suggest that ATR influences cell mechanics regulating nuclear plasticity. Here we investigate the function of ATM in relation to cell mechanics using a variety of techniques to study the mechanical properties of the cells, their plasticity and their ability to migrate across constrictions.
We found that lack of ATM activity correlates with structural alterations in the cytosol and increases cell stiffness. Moreover, defective ATM impairs 2D locomotion and interstitial migration. ATM is activated in cells subjected to deformation of the nucleus during the squeezing across constrictions in 3D migration and ATM depletion impairs survival of cells migrating inside constrictions. Altogether these observations suggest that ATM plays a role in the control of cell plasticity and migration, possibly through the regulation of cytoskeletal components.
Clara Bernardelli Different extracellular vesicles subpopulations characterize metastatic progression: qualitative and quantitative analysis of isogenic melanoma cell lines and their secreted factors The survival and proliferation of metastases is a consequence of the pre-metastatic niche PMN evolution, an abnormal, tumor growth-favoring microenvironment devoid of cancer cells. Among tumor derived secreted factors, extracellular vesicles EVs are key players in PMN establishment and facilitate organotropic metastasis.
Compared to normal melanocytes, melanoma cells produce a large quantity of EVs, that can be detected in the plasma of melanoma patients. For this reason, a full characterization of secreted vesicles subpopulations and of their cargo is necessary to understand how EVs affect PMN formation.
In this study, we demonstrated for the first time that EVs secreted by isogenic primary tumor and metastatic melanoma cell lines are quantitatively and qualitatively different, suggesting that diverse EVs subpopulations characterize metastatic progression. We also set a deep quantitative proteomics protocol to analyze the proteome of these cells and of their EVs and soluble secreted factors.
Finally, we observed that increased levels of RAB27A protein in metastatic cells do not correlate with an increased EVs secretion. Alice Bertocchi Role of the Gut Vascular Barrier in metastatic Colon Cancer Spreading of neoplastic cells from colon cancer to regional lymph nodes LN is often associated with distant recurrence. This phenomenon is probably due to a metastatic dissemination that occurs via the systemic blood circulation, rather than the lymphatic vessels.
How such dissemination is achieved is unknown. We hypothesized that the disruption of the vasculature in colon tumors can be linked to cancer cells dissemination. We identified a new marker of deranged vasculature which is expressed in primary tumors of CC patients that have developed metachronous distant metastases, independently from the presence of metastases in regional LN. Moreover, we studied vasculature disruption in a mouse model that develops spontaneous tumors mainly in the colon.
These mice showed dismantled vasculature at tumor level and they concomitantly exhibited the formation of a pre-metastatic niche at distant sites which favored the recruitment of metastatic cells. Vascular impairment can shed light on the process of colon metastases and this new marker can be used as a prognostic marker for distant recurrence.
Stefania Bertora Role of nuclear envelope protein MAN1 in nuclear organisation and maintenance of genome stability The eukaryotic nucleus is characterized by a defined spatial organization of the chromatin, which relies on the physical tethering of many genomic loci to the inner surface of the nuclear envelope, that occurs through lamins and lamin-associated proteins. Man1 is a member of a lamin-associated protein family characterized by the presence of a highly conserved chromatin-binding domain LEM.
Data obtained with yeast Man1 homolog underline the importance of this protein in different processes of the cell cycle, such as chromosome segregation, nuclear pores assembly, gene expression, chromatin organization and maintenance of genomic stability, while in animal models, the function of Man1 has been associated to the regulation of developmental signalling pathways. In this study, truncated mutants of Man1 containing the LEM domain added to Xenopus laevis cell-free extracts were shown to inhibit nuclear assembly and alter nuclear pores formation.
Moreover, Xenopus nuclei assembled with Man1 truncated fragments were characterized by defects in chromatin organization, DNA replication and accumulation of DNA damage and, as a consequence, they failed to progress through mitosis. Furthermore, mouse embryonic stem cells mESCs depleted for Man1 showed signs of spontaneous differentiation, indicating inability in the maintenance of stem cell features. Intriguingly, preliminary analysis of Man1-knockout mESCs transcriptional profile showed an alteration of gene expression at the level of pericentromeric and telomeric regions, underlining a potential link between Man1 and genomic stability of these particular regions.
In conclusion, this study illustrates the importance of Man1 in ensuring the proper chromatin organization necessary to support different cellular processes. Researchers are trying to understand how BBB is induced during development and maintained in adult life in order to develop new tools to enhance drug delivery across BBB.
Many signaling pathway that are active during BBB development are unraveled through mainly reverse genetic approach and gene knock out studies. However many target molecules or effectors of this pathway remain to be identified.
It plays a key role in arterial differentiation of the vasculature of different organs. We found that Sox17 is also expressed at high levels in brain ECs throughout embryo development and in the adult vasculature. We hypothesized therefore that Sox17 might be involved in BBB development and maintenance downstream to the Wnt pathway. In Sox17 null mice we analyzed different functional characteristics of BBB such as permeability control and specific markers expression.
Sox17 expression and signaling may be important in pathological conditions like intracranial tumors and modulation of its activity could have clinical implications and therapeutic benefits. Matteo Biancospino Functional and structural characterization of myosin VI isoforms Myosin VI is unique among the many members of the myosin superfamily.
The current understanding of the myosin VI protein does not explain how it carries out these diverse processes, as functional mechanistic studies are lacking. Alternative splicing in the tail region generates myosin VI molecules with different features myosin VIshort and myosin VIlong but little is known about the impact of the variable region on physiological and pathological functions of myosin VI. In this study, we have analysed the myosin VI isoforms from a molecular and a functional perspective.
Its presence or absence determines the target selectivity of myosin VIshort and myosin VIlong isoforms, acting like a molecular switch that regulates their functional involvement in migratory or endocytic pathways, respectively.
Thus, we can provide for the first time a mechanistic explanation of why the various isoforms show different localization and function i. Importantly, cancer cell lines selectively expressing the myosin VIshort isoform exhibit severe migration defects when myosin VI is knocked down. Therefore, the development of novel therapeutic strategies that specifically target the CSCs population could be the key to achieve an effective cure for breast cancer.
We used a lentiviral microRNA library to perform a functional whole-genome screening based on the self-renewal ability of CSCs on a breast cancer cell line. The screening yielded to 20 candidate microRNAs selected as potential modifiers of selfrenewal.
A proof-of-principle validation revealed that 6 out of 10 tested cloned, confirmed their effects even when analyzed as single clones, underlining the potentiality of the whole-genome phenotype screening. We focused our attention on the two most promising candidates and, in order to search for the mechanism through which these microRNAs exert their function, we performed an RNA-seq analysis. We revealed that one microRNA in particular was able to control independently different pathway related to self-renewal, migration and proliferation, suggesting that this miRNA could effectively act at multiple levels to silence the selfrenewal potential of cancer stem cells and, likely, inhibit the proliferation and migratory ability of the tumor, too.
Heide-Marie Binder Sleeping Beauty finds cooperating partners of oncogenic Myc Myc triggers a transcriptional program inducing hyper-replication and proliferation but also tumor suppressive mechanisms like apoptosis. Therefore, Myc dependent tumors display high selective pressure to accumulate secondary mutations blocking these tumor-suppressive pathways.
In order to find novel cooperating partners of Myc leading to transformation we applied a conditional Sleeping Beauty transposon-based mutagenesis screen in vivo. The genomic sequences immediately adjacent to integrated transposons of lymphomas were enriched by ligation-mediated PCR and were sequenced with a multiplexed approach.
Based on published Brett et al. Thus, IRSp53 is essential for coordinating these activities in vitro and in cells in culture. In the second part of this thesis, an IRSp53 role in the polarity program was investigated. We speculate that IRSp53 might be involved in the trafficking of the apical protein podocalyxin, we are currently investigating the molecular mechanism responsible for this putative role. Margherita Bodini Genomics of treatment response in acute myeloid leukaemia Acute Myeloid Leukemia AML is a cancer of the myeloid lineage of blood cells.
The molecular basis of relapsing disease is still unclear and the few identified predictive factors has small predictive power. Currently, chemotherapy induction treatment consists in the administration of mainly three drugs fludarabine, cytarabine, and idarubicin.
In this thesis, endowed of the NGS technologies advancement, we decided to delineate the possible process of relapse formation in order to be able in the future to predict which patients are more susceptible to relapse. Federico Boem A matter of style. How map thinking and bio-ontologies shape contemporary molecular research The aim of this thesis is to provide an epistemic analysis of the transformations occurring in contemporary biological research by considering the relation between molecular biology and computational biology.
In particular, I will focus on bio-ontologies, as the tool which incarnates at best the new face of biomedical research. Such a choice is not arbitrary. By appealing to the notion of style of reasoning and way of knowing, I will show that bio-ontologies exemplify the rise and success of map thinking as the signature of a new way of doing molecular biology, while the theoretical tenets, established more than 30 years ago, still maintain their epistemic prominence.
This is neither to say that experimentalism will disappear from science, nor that the experiments power will be diminished but rather that experiments will have a new role in the architecture of scientific efforts, precisely because of the increasing importance of classificatory approaches.
Therefore, such a transition within biomedical research is indeed radical and profound but it does not involve paradigm shifts but rather a change in the practice. In this sense, it is a matter of style. Such mutations give rise to the so-called Leukemia Initiating Cell LIC , characterized by enhanced self-renewal and impaired differentiation.
The molecular mechanisms underlying this transition are still poorly understood but they are likely to be critical to understanding the leukemic stem cell LSC biology. We thus hypothesized that enforced quiescence might be critical to maintain the transformed clone during both the pre-leukemic and the leukemic phase.
Chromosome segregation is carried out by the mitotic spindle, which captures and pulls sister chromatids towards the opposite poles. Anaphase starts when the correct bipolar attachment is achieved. Chromosomes migrate evenly to the two daughter cells, both inheriting the same genetic material.
The presence of unattached kinetochore at anaphase onset is dangerous, since it may lead to unbalanced ploidy of daughter cells, with severe consequences for their survival. For this reason, improperly attached chromosomes activate the mitotic checkpoint that arrests cell division before anaphase. Cells can maintain an arrest for several hours but eventually will resume proliferation, a process we refer to as adaptation.
Whether adapting cells bypass an active block or whether the block has to be removed to resume proliferation is not clear. Likewise, it is not known whether all cells of a genetically homogeneous population are equally capable to adapt. Here, we show that the mitotic checkpoint is operational when yeast cells adapt and that each cell has the same propensity to adapt.
Our data provide a quantitative framework for understanding how cells overcome a constant stimulus that halts cell cycle progression. When overexpressed, Myc binds to almost all open promoters but only regulates specific subsets of genes. In order to analyse the DNase-seq footprinting data in our systems, we developed a novel pipeline that carries out step-by-step analysis of the raw DNase-seq data, and outputs DHS and TF footprints.
We overlapped genome wide the footprints identified by the pipeline with matches of a PWM library, obtaining a list of footprinted PWMs. We first applied a single feature classifier assessing the performance of each of the PWMs one by one, and we found that single PWMs only provided a limited classification of the gene subsets. We then turned to a random forest classifier that considers combinations of PWMs as features. The organ location is critical, both for neurological consequences and therapeutic intervention, which is exclusively surgical to date.
The cause of CCM can be either sporadic or genetic. Here we describe a novel murine model of the disease that develops after endothelial cell-selective ablation of the CCM3 gene in newborn mice and that we use to investigate the molecular mechanisms behind the development of CCM. We report enhanced transcription activity of b-catenin in CCM3-knockout endothelial cells in in vitro and in vivo models and we demonstrate that such activation is critical at early stage of the pathology development.
In particular, we found that b-catenin is fundamental to trigger the process of endothelial-to-mesenchymal transition EndMT , which has been previously demonstrated to be crucial for CCM development. Noteworthy, the activation of b-catenin pathway results Wnt-independent, while it correlates with the impaired state of endothelial cell-to-cell junctions typical of vessels developing CCM lesions. We also show that a pharmacological screening of a panel of drugs targeting b-catenin signaling revealed the NSAIDs sulindac sulfide and sulindac sulfone as potent inhibitors of this signaling pathway in endothelial cells.
Moreover, we found that sulindac sulfide and sulindac sulfone are able to attenuate b-catenin transcription activity and to significantly reduce the number and dimension of vascular lesions in the central nervous system of mice with endothelial cell-specific CCM3-knockout. These NSAIDs thus represent pharmacological tools for inhibition of the formation of CCM3 vascular lesions, particularly with a view to patients affected by the genetic variant of CCM, who continue to develop new malformations over time.
Serena Buontempo Functional dissection of Ezh2 during neuraldifferentiation Epigenetic control of developmental genes is a key mechanism to achieve a time-regulated expression during differentiation as demonstrated to date in several differentiation pathways. In embryonic stem cells these developmental genes are marked by histone H3 lysine 4 H3K4me3 and histone H3 lysine 27 H3K27me3 trimethylation. The preservation of these bivalent marks is essential for proper time-regulated expression.
It has been shown however that H3K27me3, catalyzed by Ezh2 within Polycomb Repressive Complex 2 is critical for the entire process of differentiation. Thus during neural differentiation, Polycomb targets have been shown to be dynamic with new genes acquiring or losing the mark at each step of neural differentiation.
However most of the efforts were done on lineage commitment while Ezh2 role in the late phases of neural differentiation is less studied. In this work the role of the epigenetic axis focusing on Ezh2 loss in post-mitotic neurons was dissected. To this end, after ES cells derivation from Ezh2 conditional mice, a neural differentiation protocol was used to produce a homogeneous population of glutamatergic post-mitotic neurons. The lack of Ezh2 does not impair survival and development of neurons in culture.
Although genome-wide transcriptomic approach revealed that Ezh2 is needed in post-mitotic neurons to maintain proper gene expression of genes involved in glutamate transmission, in particular our analysis shows an imbalance in glutamate receptors subunits expression. Thus our data suggest that post-mitotic neuronal maturation might be epigenetically controlled through Ezh2 regulation of glutamatergic pathway. Claudia Burrello Gut microbiota crosstalk with conventional and non-conventional T cells: a game of many players The presence of microbial commensals in the gut requires the establishment of a complex network of reciprocal interactions between the microbiota and the host immune system to allow nutrient absorption while preventing undesired mucosal immune responses.
Despite these homeostatic mechanisms, during intestinal inflammation alterations of the microbiota composition, namely dysbiosis, trigger abnormal immune responses. Here, we aimed at investigating the functional crosstalk between gut microbiota and the mucosal immune system during inflammation and upon induction of microbial dysbiosis. Moreover, during intestinal inflammation, the experimental manipulation of the microbiota community through Faecal Microbiota Transplantation FMT reduces colonic inflammation and initiates the restoration of intestinal homeostasis through the induction of IL production by immune cells.
We report that the exposure to mucosa-associated microbiota drives iNKT cell pro-inflammatory activation, inducing direct pathogenicity against the intestinal epithelium. Collectively, we provided solid evidence that a strict crosstalk between the gut microbiota and the intestinal conventional and non-conventional T cells exists. Antibiotic-associated dysbiosis has immunostimulatory functions. Moreover, FMT can therapeutically control intestinal experimental colitis and this poses FMT as a valuable therapeutic option in immune-related pathologies.
In addition, we generated fundamental knowledge about the pathogenic functions exerted by human intestinal iNKT cells upon the interaction with mucosa-associated microbiota communities. Specifically, nowadays the components of the uPA-system are well-characterised determinants for the prognosis of breast cancer. The regulation of the gene expression of the uPA-system components is very complex and depends on a plethora of stimuli acting both at transcriptional and post-transcriptional level.
The uPA-system components are often over expressed in breast cancer but the detailed molecular mechanisms regulating the expression are still to uncover. We experimentally validated the role of miR as negative regulator of the expression of the three uPA-system components using MDAMB- , a triple negative breast cancer cell line.
Microarray experiments, performed to characterise the global transcriptome changes induced by miR in MDA-MB cells, showed that miR- down regulates also the expression of desmoplastic reaction-related genes underlining a possible role of miR in regulating tumour-associated genes. Notably, most of the identified miR target genes were found indeed to be associated with poor clinical outcome in breast cancer.
Functional studies carried out in MDA-MB cells suggested that miR might modulate cell proliferation, even if this effect was not confirmed in vivo. In order to better define the functional role of miR, we generated a miR deficient mouse model, taken advantage of the zinc finger nuclease technology. Overall these data identify, for the first time, a single microRNA that is able to down regulate the expression of the three main components of the uPAsystem together with desmoplastic reaction and breast cancer prognosis-related genes, thus representing a new potential player in the pathogenesis of breast cancer.
Even though animal studies have revealed a lot of its unfolding, the fine regulation of cellular differentiation trajectories that characterizes humans has become only recently open to experimental tractability, thanks to the development of organoids, human cellular models that are able to recapitulate the spatiotemporal architecture of the brain in a 3D fashion. Here we first benchmarked human brain organoids at the level of transcriptomic and structural architecture of cell composition along several stages of differentiation.
Then we harnessed their properties to probe the longitudinal impact of GSK3 on human corticogenesis, a pivotal regulator of both proliferation and polarity, that we revealed having a direct impact on early neurogenesis with a selective role in the regulation of glutamatergic lineages and outer radial glia output. Moreover, we spearheaded the use of organoids for regulatory toxicology through the study of Endocrine disrupting chemicals EDC , pervasive compounds that can interfere with human hormonal systems.
Early life exposure to EDC is associated with human disorders, but the molecular events triggered remain unknown. We developed a novel approach, integrating epidemiological with experimental biology to study the mixtures of EDC that were associated with neurodevelopmental and metabolic adverse effects in the biggest pregnancy cohort profiled so far. Our experiments were carried out on two complementary models i human fetal primary neural stem cells, and ii 3-dimensional cortical brain organoids and we identified the genes specifically dysregulated by EDC mixture exposure, unravelling a significant enrichment for autism spectrum disorders causative genes, thereby proposing a convergent paradigm of neurodevelopmental disorders pathophysiology between genetic and environmental factors.
Finally, while EDCs are everywhere, their impact on adverse health outcomes can vary substantially among individuals, suggesting that other genetic factors may play a pivotal role for the onset of the disorders. We took advantage of organoids multiplexing to recapitulate, at the same time, neurodevelopmental trajectories on multiple genetic backgrounds, and showed that chimeric organoids preserved the overall morphological organization and transcriptomic signatures of the ones generated from single lines.
In conclusion our work shows the possibility to perform population level studies in vitro and use the deep resolution of molecular biology to dissect key aspects of human neurodevelopment. Planar divisions occurring with the spindle within the epithelial plane enlarge sheets and tubules, while asymmetric cell divisions with the spindle aligned to the apico-basal polarity axis sustain differentiation programs. Several pathways have been involved in establishing correct spindle orientation, both in cultured cells and in vivo.
My PhD project focused on the molecular mechanisms underlying the spindle orientation function of Afadin, and on the relevance of NuMA phosphorylation by Aurora-A for spindle orientation. Furthermore, we 2 showed that loss of Afadin impairs correct cystogenesis of Caco-2 cells, suggesting that it plays essential functions in epithelial planar cell divisions.
It was reported that the activity of the mitotic kinase Aurora-A is required for correct spindle orientation in human cells in culture, and that NuMA is among its phosphorylation targets. However, whether NuMA is phosphorylated directly by Aurora-A and how molecularly its kinase activity affects spindle orientation was still unknown when we started our studies. Experiments in vitro and in cells showed that Aurora-A phosphorylates directly three serine residues on the C-terminus of NuMA, and mutation of Ser into alanine recapitulates the aberrant polar accumulation of NuMA and the spindle orientation defects observed upon Aurora-A inhibition.
Thus we concluded that phosphorylation on Ser of NuMA by Aurora-A controls 3 NuMA distribution between the spindle poles and the overlying cortex, and allows proper spindle orientation. Intriguingly, Ser lies within a previously characterized MT-binding domain.
In vitro co-sedimentation and bundling assays revealed that the binding affinity of NuMA for MTs is unaltered by Aurora-Amediated phosphorylation, suggesting that unphosphorylated NuMA accumulates at spindle poles via a receptor other than MTs.
Alessandro Carugo Dissection of the molecular pathways involved in pancreatic cancer initiation and progression with a novel in vivo approach Abstract not available Lucia Casoli Myc-dependent transcriptional programs in mammary epithelial cells The transcriptional programs governing the decision of mammary epithelial stem and progenitor cells to self-renew or differentiate are still not completely outlined. Previous evidences pointed out the role of Myc, and in particular of its repressive activity with Miz1, in these processes.
Since the presence of stem-like cells within the tumor, the so-called cancer stem cells CSCs , is now considered crucial for tumor initiation and maintenance, clarifying this aspect of Myc biology could be relevant in understanding its contribution to the genesis of breast cancer. Here, we used RNA-seq technology to profile the transcriptional programs regulated by Myc in two different settings.
First, we studied immortalized mammary epithelial cells, in which we induced either Myc loss or gain of function. In this setting, Myc contributed to the positive and negative regulation of different sets of genes. Activated genes are involved in proliferation, metabolism, ribosomal biogenesis, mitochondrial organization, chromatin modification, RNA processing and modification.
Repressed genes, on the other hand, were mainly involved in lysosome and vesicle-mediated transport, angiogenesis, cell death, extracellular matrix interaction, cell adhesion regulation, epithelial development and morphogenesis. Second, we studied the effect of Myc activation in mammosphere cultures, which provide a measure of stem cell activity.
We demonstrate that Myc, when overexpressed, is able to promote self-renewal of mammary epithelial stem cells, as assessed by increased mammosphere expansion, and confirmed by mammary gland reconstitution assays in vivo. This activity of Myc is in part dependent on the interaction with the co-repressor protein Miz1, since the Myc mutant VD hereby Myc VD , impaired in Miz1 binding, is defective in promoting self-renewal.
Overexpression of Myc in mammospheres was associated with the de-regulated expression of about three thousand genes, with similar numbers of up- and down-regulated genes. Surprisingly, the overlap between the groups of regulated genes in those mammary epithelial cells in adhesion or grown as mammospheres was limited, illustrating the context-dependency of Myc-dependent responses. Thus, transcriptional repression via Miz1 may constitute one of the mechanisms through which Myc sustains mammary epithelial cell selfrenewal.
We are currently setting a functional screen, among genes repressed in a Miz1-dependent manner, to identify those that are critical in this process. Our study shall shed light on the mechanisms through which Myc regulates self-renewal in mammary epithelial stem and progenitor cells. Understanding this could be crucial in order to clarify the physiopathological roles of Myc in the mammary gland. These multiprotein complexes are key repressive chromatin factors that regulate cellular differentiation during development, contributing to the correct establishment of lineage-specific transcriptional programs.
Moreover, they represent key factors of proliferation and deregulation of their levels and activity have been linked to the onset and development of several human cancers. Recently, gain of function heterozygous EZH2 mutations have been discovered in non-Hodgkin lymphomas and melanomas. Very little is known about this mutated enzyme, therefore the aim of my thesis is trying to unravel the tumorigenic mechanisms underlying these mutations.
To understand a general oncogenic role for this mutated enzyme, we used MEF as an alternative, simpler model system. We observed increased deposition of H3K27me3 without any relevant transcriptional alteration at steady state, confirming our results also in lymphoma cell lines. To investigate a cooperative transcriptional deregulation for mutant EZH2, we then subjected MEFs to three different stimuli starvation, myc upregulation and reprogramming to pluripotency. Since we found this to be true only during cell-fate transition, we proposed a model in which the levels of the H3K27me3 are increasingly deposited where the mark is already present at steady state.
This could be relevant in lymphomas, impeding centroblasts differentiation and resulting in tumorigenesis in the presence of concomitant oncogenic mutations. This observation could shed light on the molecular mechanisms underlying lymphomagenesis in patients. Claudia Cella Development of biodegradable nanoparticles for targeting Tumor Associated Macrophages: synthesis, investigation of the role of the surfactant and surface decoration in complex media Tumor Associated Macrophages TAMs are involved in cancer proliferation, thus strategies to deplete them represent promising tools for chemotherapy.
Pharmacological agents with multiple activities such as curcumin and RNA interference have been proposed; however their employment in therapeutics has been limited because of low systemic bioavailability. Accordingly, this thesis described as an innovative therapeutic approach for cancer treatment the development of polymeric nanoparticles NPs able to i increase pharmacokinetics properties of biomacromolecules and poor water soluble drugs, and ii guarantee TAMs specific targeting.
Different synthetic parameters have been taken into consideration, with particular focus on the surfactant role. NPs with solid or core-shell structures, whose size was tailored between and nm, were obtained and a thorough characterization has been performed, with the help of innovative techniques such as single particle optical extinction and scattering SPES method.
Strategies for surface decoration with the employment of D-mannose as specific molecule to guarantee TAMs recognition were proposed. By performing a whole genome analysis of DNA replication dynamics occurring upon oncogene activation, I discovered that oncogene activation alters DNA replication by increasing replication fork speed and fork stalling, while decreasing the frequency of replication initiation.
This is accompanied by a prompt DDR activation. As cells approach senescence the frequency of initiation increases, the level of fork stalling and fork speed decreases. Oncogene activation leads to DNA replication stress mainly at fragile sites and since telomeres resemble fragile sites, I then demonstrated that oncogene activation impairs telomere replication, by increasing fork stalling at telomeres.
This is accompanied by increased fragile telomeres, stochastic telomeric attrition and persistent telomeric DDR. These results revealed a novel link between oncogene activation and telomere dysfunction, refining the model underlying OIS establishment. Beyond their toxicity, ROS are essential second messengers mediating mitogenic signalling. Upon oncogene activation, NOX4 pharmacological inhibition blocks ROS production, resulting in fork speed reduction and differential regulation of local replication origin initiation.
Luca Chiapperino From consent to choice: the ethics of empowerment-based reforms The aim of my thesis is twofold. What I define as EBRs will have in fact different implications for the various stakeholders involved in their development and implementation. Empowered citizens within EBRs will have access to and will be required to manage an unprecedented amount of information regarding their health conditions.
Factors such as genetic and biological makeup, life-style behaviours and environmental exposures will be increasingly used by both citizens and professionals to identify treatment options, to target developing diseases, and to adopt preventive measures for future illnesses.
Among the effects that this personalising vision of healthcare is likely to foster, it is thus worth emphasizing how the nature and scope of individual responsibility for health will be affected by this paradigm shift, and how this future scenario can be made an ethically desirable one.
In my thesis, I therefore identify the range of normative exercises entailed in EBRs, and I present a normative analysis of empowerment aiming at highlighting the distinctive ethical aspects of this approach. Second, the goal of my thesis is to explore how the normative theorization of empowerment proposed above can accommodate one of the most pressing societal implications of epigenomics.
Namely, its burdening of individual responsibility for health. On the one hand, I argue that novel approaches to prevention, diagnosis and treatment brought about by epigenomics are a fundamental tenet of the personalization project at the basis of what I call EBRs. In this respect, epigenome-based healthcare is thus likely to foster controversies similar to other epistemic endeavours of personalized medicine e. Rather than limiting societal appraisal of epigenomics to the danger of burdening individual responsibility for health, I argue that epigenetic knowledge may become pivotal in fleshing out social and environmental influences inherently affecting individual health.
Sufficiently valid, reliable and actionable epigenetic knowledge may in fact orient individual choice across the spectrum of environmental and lifestyle exposures determining health, thus championing epigenomics with the potential of serving the empowering aims fleshed out throughout this work. The road connecting the constitution of an empowered citizenship in healthcare, and the societal appraisal of epigenomics can be regarded as a two-way road.
There is in fact a possibility that empowerment and epigenomics may respectively shape their normative and epistemic dimensions in the future of healthcare. It is thus towards the identification of the possible challenges and opportunities that this synergy may bring about that the theoretical attention of this work is devoted.
Ramveer Choudhary Mechanisms controlling the integrity of converging forks during replication termination During S phase,natural fork pausing elements including replication termination zones TERs and transcribed genes, can easily lead to!
However, the key factors and pathways protecting the integrity of CFSs are not well understood. In order to elucidate these mechanisms, we used yeast as a model system, and combined genetic and genomic studies aimed at identifying fragile sites genome wide. We found that top2 contributes to relieve the topological stress and extensive fork pausing during Termination at TERs.
We also observed that fragility at these sites is greatly enhanced in checkpoint mutants Tel1ATM1 and Mec1 ATR1 , likely due to their inability to uncouple transcription from gene gating, thus leading to topological stress in front of replication forks. TER fragility in checkpoint defective! We show thatprogrammed fork pausing and resolution of R loops are key processes for TER integrity.
Taken together, our data strongly suggests that, the replication checkpoint, Sen1 and Rrm3, coordinate replication termination to prevent accumulation of unsolved topological constrains and premature recruitment of Condensins andTop2. Cecilia Claudi Polo-like kinase Cdc5 contributes to mitotic spindle elongation via the kinesin-5 motor protein Cin8 Proper chromosome segregation requires an orderly sequence of events, whereby spindle elongation follows the dissolution of sister chromatid linkages.
The mitotic spindle is a sophisticated and complex machinery built of microtubules, microtubule associated proteins and motor proteins. Proper spindle function requires that microtubule dynamics are stabilized at anaphase. This change in microtubule dynamics is dictated by a shift in the balance of kinase and phosphatase activities in favor of the phosphatases.
The finding that cells simultaneously lacking the polo-like kinase Cdc5 and the phosphatase Cdc14 cannot progress through anaphase albeit having cleaved cohesin due to defects in spindle elongation, challenges the view of mitotic exit as a time for protein dephosphorylation. We show that besides being dephosphorylated by Cdc14, Cin8 is also phosphorylated by Cdc5 on residues S and S, and that this phosphorylation is crucial for the function of the kinesin in anaphase spindle elongation.
The finding that Cin8 is simultaneously a substrate of a kinase and a phosphatase sheds light on the complexity of mitotic exit regulation. Since it appears that phosphorylation and dephosphorylation events are equally important to the point that kinases and phosphatases cooperate to regulate the same substrates, the view of mitotic exit as the realm of phosphatases is dismantled and the continuous need for single molecule studies in addition to global analyses investigation is put forward. Agnese Collino Characterization of genetic and epigenetic modifications in a model of inflammation-driven cancer Chronic inflammation is causally associated to many types of tumor, and has been recently acknowledged as a cancer hallmark.
Nevertheless, whether inflammation has an intrinsic mutagenic potential is still not directly proven or understood from a mechanistic point of view. Sirtuins primarily serve as antioxidants in redox signaling. Furthermore, in the mitochondrial inter-membrane space, SIRT5 deacetylates cytochrome c Schlicker et al. HDAC-involved metabolic regulation. HDACs regulate metabolism mainly including glycometabolism, lipid metabolism, amino acid metabolism and redox.
Panobinostat-induced downregulation of meiotic recombination 11 homolog MRE11 enhances radio-sensitization of bladder cancer cells by promoting MRE11 ubiquitination that relies on the upregulated E3 inhibitor of apoptosis protein 2 cIAP2 Nicholson et al.
Entinostat represses checkpoint signaling during replication stress. Entinostat also induces the incorrect incorporation of NTPs and metabolites during the induction of checkpoint kinase inactivation, which can result in mitosis catastrophe Goder et al. Therefore, CHK inhibitors might be designed to prevent this event from occurring. This process is closely associated with chromatin remodeling in response to DNA damage Bao et al. Besides, a number of other histone modifications are also actively involved in these DNA repair pathways, but are beyond the scope of this review Cao et al.
Suffice to say that the multiple sites of H3 and H4 acetylation are not absolutely related to checkpoint activation because the conversion of lysine to other amino acids can still activate checkpoints Robert et al. Cell Cycle Cell cycle dysregulation is a central hallmark of oncogenesis; as such, cell cycle regulators are considered promising targets for cancer treatment. HDACs are often involved in cell cycle checkpoints. Spindle assembly checkpoint SAC is involved in regulating mitosis.
SIRT2 is strongly associated with mitosis exit Dryden et al. Combination of the Aurora A kinase inhibitor alisertib with romidepsin causes dose-dependent cytotoxicity of lymphoma cells Zullo et al. Namely, SIRT7 indirectly modulates pmediated cell cycle arrest by elevating p53 activity. Apoptosis Apoptosis is a physiologically programmed cell death pathway that is essential for the maintenance of organismal homeostasis.
Apoptosis is controlled by the B-cell lymphoma 2 Bcl-2 family of proteins, which includes both pro-survival and pro-apoptotic proteins that control cell fate Singh et al. Regarding the intrinsic apoptotic pathway, the Bcl-2 interacting mediator of cell death Bim , a Bcl-2 homology 3 BH3 -only proapoptotic protein, is upregulated by depsipeptide via FoxO1 acetylation Yang et al. Panobinostat elevates Sry-box transcription factor 7 SOX7 expression and suppresses lung cancer cell proliferation.
Mechanically, SOX7 triggers apoptosis by preventing Bim from proteasome-mediated degradation Sun et al. Histone deacetylase inhibitors treatment also affects the anti-apoptotic members of the Bcl-2 family. Specifically, depsipeptide induces apoptosis by decreasing the expression of pro-survival factors Bcl-2 and B-cell lymphoma-extra-large Bcl-xL Adams and Eischen, ; Adams et al. The HDAC6-selective inhibitor ricolinostat exerts pronounced anti-lymphoma effects both alone and in combination with the alkylating agent bendamustine, by impairing the activation of caspase 8, -9, -3, and the Bcl-2 family Cosenza et al.
Myeloid cell leukaemia 1 Mcl-1 is an E3-bound, anti-apoptotic protein that is involved in mitotic arrest Senft et al. Acetylation of p53 at K upregulates apoptotic peptidase activating factor 1 Apaf-1 in the mitochondria Yun et al. In summary, HDACi promote apoptosis via the intrinsic mitochondrial pathway, decreasing the expression of key anti-apoptotic factors eg.
Degradation System The modulation of protein degradation is of critical importance for cell function. Protein degradation occurs via two major pathways: the ubiquitin-dependent proteasome pathway and autophagy system.
Autophagy Autophagy is a degradation process whereby autophagosomes engulf and recycle nutrient sources in response to energetic demands and organelle turnover Mizushima et al. Autophagy can be effectively promoted by HDACs. HDAC10 inhibition results in increased sensitivity to cytotoxic reagents Oehme et al. The SIRT1 and -2 inhibitor tenovin-6 activates p53 and seems to be a specific regulator of mitochondrial acetylation Lain et al.
In response to serum starvation or oxidative stress, SIRT2 inhibition induces acetylated FoxO1 to locate in the cytoplasm, accelerating autophagy through interaction with ATG7 Zhao et al. The nutrient-sensor mammalian target of rapamycin mTOR negatively modulates downstream Unclike autophagy activating kinase 1 ULK1 that is involved in the non-transcriptional autophagic pathway.
Proteasome-Dependent Degradation In addition to autophagy, proteasome-dependent degradation is also critical for cell function. HDACs target various E3s to affect basal cellular function. For example, panobinostat upregulates the E3 cIAP2 that causes the ubiquitination and proteasomal degradation of MRE11, elevating cellular sensitivity to chemoradiation Nicholson et al.
HDAC6 also modulates aggresome formation and the clearance of polyubiquitinated and misfolded proteins Kawaguchi et al. In terms of therapeutic development, suppressing the aggresome pathway results in the accumulation of misfolded proteins, causing autophagy-associated DNA damage and apoptosis of cancer cells Rodriguez-Gonzalez et al.
However, long-term treatment with BTZ leads to drug-resistance in most patients. Low concentrations of HDACi combined with BTZ can downregulate anti-apoptotic proteins and upregulate pro-apoptotic proteins, thus accelerating cell death Dai et al. Epithelial-Mesenchymal Transition, Cancer Stem Cells, and Senescence Epithelial-mesenchymal transition EMT is characterized by the loss of the tight intercellular connections normally found in epithelial cells that then undergo cytoskeleton rearrangement and adopt the mesenchymal cell phenotype, which is associated with migration.
Cancer stem cells are hard to eradicate and prone to drug-resistance. HDAC3 interacts with p53 and forms complexes with tumor antigens melanoma antigen family A2 MAGE-A2 , establishing the resistance of melanoma cells to chemotherapeutic agents Monte et al. In refractory and recurrent leukemia, HDAC8-selective inhibitor significantly restores acetylation and p53 activity, inducing apoptosis of AML cells but not of normal hematopoietic stem cells Qi et al.
Sirtuins are also closely involved in aging-related oncogene expression. These repressive functions of L1 highlight the protective roles of SIRTs on genome stability through preventing retrotransposition events. Overview of HDAC-involved biological functions and therapeutic targets. An overview of HDAC-involved biological functions including transcription, metabolism, oxidative stress, redox, protein degradation, cell cycle, DNA damage repair, apoptosis, angiogenesis, EMT, immunity, and stemness.
There diverse functions could establish single or synergistic therapeutic targets. HDAC Inhibitors in Cancer Therapy In the s, sodium butyrate was discovered to transform red leukemia cells into normal cells, and to resynthesize hemoglobin. This process was accompanied by strong histone hyperacetylation, and resulted in the discovery of the first HDACi Ginsburg et al. In Tsuji et al. A number of natural inhibitors have since been extracted from fungi, marine life, and plants that contain sulfur, polyphenol, flavonoid, terpenoid, selenium, and other organic molecules Newkirk et al.
Current clinical trials involving the use of HDAC inhibitors to treat cancer. HDACi can significantly attenuate tumor burden by limiting tumor growth and restraining aberrantly proliferated vessels Guerriero et al. HDACi can also induce DNA damage, cell cycle arrest, apoptosis and autophagy to promote cancer cell death mentioned above. Activation of the immune response by HDACi could also be an effective innate method to prevent cancer relapse when administered in a regimen with immunotherapeutic Guerriero et al.
Upon immune checkpoint blockade, TMP combined with an anti-programmed cell death-1 PD-1 regimen could significantly reduce the tumor volume and induce a durable response in breast cancer Guerriero et al. Myeloid-derived suppressor cells MDSCs suppress T-cell functions and promote tumor metastasis via the formation of an immunosuppressive tumor microenvironment Azzaoui et al.
In many clinical trials, SAHA has proven effective against advanced and refractory tumors, alone or in combination with other inhibitors. Both panobinostat and belinostat are classified as hydroxamic acids. Most SIRT inhibitors still remain in the preclinical stages. So far, only nicotinamide vitamin B3 has been used to treat cancer in clinical trials e.
Nicotinamide has shown a potential role in inhibiting non-melanoma skin cancers that are principally generated by UV Chen et al. Compared to other HDACi, nicotinamide exhibits the most catalytic sites: it is predominantly sensitive to acetylation sites in nuclear proteins that are involved in diverse biological processes Scholz et al. Application of Selective Inhibitors and Combination Therapy With the development of HDACi, numerous clinical trials are ongoing or completed currently for cancer therapy.
Many HDACi have already been approved for hematological malignancies and lymphomas, while clinical studies are ongoing for refractory, advanced and recurrent solid tumors Table 4. HDACi with multiple targets have also been developed and tested in clinical trials, such as the dual HDAC and phosphoinositide-3 kinase PI3K inhibitor CUDC also called fimepinostat , which has been reported to inhibit Myc transcriptional expression and reduce Myc-mediated proliferation of multiple cancer cell lines Pei et al.
Clinical trials investigating the single-agents or combined therapies in HDACi and other anti-neoplastic drugs. Combination drugs can inhibit tumorigenesis from different aspects. Hydroxychloroquine HCQ is a common autophagy-targeting reagent that has been used in clinical research Mahalingam et al.
The overall survival OS and progression-free survival PFS are common and quite important indicators in the clinical trials. The median PFS reaches And patients who had received previous regimens such as immunomodulatory drug and bortezomib, median OS was only Limitations of HDACi However, in a randomized, double-blind and placebo-controlled phase III trial, vorinostat did not improve OS and could not be recommended as a therapy as a second-line or third-line drug for patients with advanced malignant pleural mesothelioma Krug et al.
The overall response to vorinostat in this study was significantly lower than reported in a previous study Olsen et al. Besides, in a phase II trial, mocetinostat did not reverse chemoresistance in patients with previous gemcitabine-resistant leiomyosarcoma and could not significantly prolong the median PFS of patients Choy et al.
However, there are also several reports demonstrating an indirect interaction between SIRT7 and p53 Barber et al. Thus, it is urgent to develop specific SIRT inhibitors for cancer therapy according to reasonable mechanisms. It is worth noting that SIRT activators and sirtuin-activating compounds STACs have been developed and studied in clinical trials to investigate their anti-aging, anti-inflammatory and metabolic regulatory effects Howitz et al.
Besides, most of clinical trials of HDACi have reported many adverse effects, including bleeding caused by different grades of thrombocytopenia, susceptibility to infection caused by neutropenia, anemia caused by hemoglobin reduction, arrhythmia, myocardial hypertrophy, neurotoxicity, and gastrointestinal toxicity such as nausea, vomiting, fatigue, diarrhea as well as electrolyte disturbance such as hypophosphatemia and hyponatremia.
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